Characterization of a novel multidomain CE15-GH8 enzyme encoded by a polysaccharide utilization locus in the human gut bacterium Bacteroides eggerthii

Bacteroidetes are efficient degraders of complex carbohydrates, much thanks to their use of polysaccharide utilization loci (PULs). An integral part of PULs are highly specialized carbohydrate-active enzymes, sometimes composed of multiple linked domains with discrete functions—multicatalytic enzymes. We present the biochemical characterization of a multicatalytic enzyme from a large PUL encoded by the gut bacterium Bacteroides eggerthii. The enzyme, BeCE15A-Rex8A, has a rare and novel architecture, with an N-terminal carbohydrate esterase family 15 (CE15) domain and a C-terminal glycoside hydrolase family 8 (GH8) domain. The CE15 domain was identified as a glucuronoyl esterase (GE), though with relatively poor activity on GE model substrates, attributed to key amino acid substitutions in the active site compared to previously studied GEs. The GH8 domain was shown to be a reducing-end xylose-releasing exo-oligoxylanase (Rex), based on having activity on xylooligosaccharides but not on longer xylan chains. The full-length BeCE15A-Rex8A enzyme and the Rex domain were capable of boosting the activity of a commercially available GH11 xylanase on corn cob biomass. Our research adds to the understanding of multicatalytic enzyme architectures and showcases the potential of discovering novel and atypical carbohydrate-active enzymes from mining PULs.

Use of carbonized corn cob biomass to reduce acidity of residual frying oil

The objective of this work was to evaluate the ability of CCC as an adsorbent material for the acidity removal of RFO, aiming at the application of the oil in biodiesel production. For that, a RCCD was used for FFA removal by applying the CCC and CAC for comparative purposes. In the RCCD removal assays the effect of the Temperature, Agitation and Mass factors were assessed over acidity removal of the oil. Under the best conditions from RCCD, an evaluation of adsorption kinetics was performed, wherein it was observed the equilibrium was reached within 4 h, for the CCC. Also, the influence of the adsorbent dosage was performed. It was verified that 4 g was sufficient to allow the system to reach the maximum FFA removal. Overall, the CCC presented results approximately twice as high than those obtained by the CAC, mainly due to the pore size distribution which led to a “molecular sieving effect” for the CCC adsorbent. It allowed the major diffusion of the FFA molecules inside its narrow-distributed pores, whereas the CAC with a wider pore distribution (up to 260 Å) resulted in the larger molecules competition for the active sites inside the porous structure. The adsorbents’ characterization also evidenced that CCC adsorbent presented a higher content of oxygenated groups in its surface which acted as potential active sites for the FFA molecules resulting in an enhanced adsorbent-adsorbate affinity. Lastly, the wastes generated in the adsorption experiments, were evaluated as to their calorific power resulting in a value of 31,933 J g-1, suggesting that it could be further used for energetic purposes, such as a solid fuel for boilers and furnaces to generate thermal energy. Based on these results, the CCC stands out as a promising material for RFO acidity removal.

Multimodular fused acetyl–feruloyl esterases from soil and gut Bacteroidetes improve xylanase depolymerization of recalcitrant biomass

Background Plant biomass is an abundant and renewable carbon source that is recalcitrant towards both chemical and biochemical degradation. Xylan is the second most abundant polysaccharide in biomass after cellulose, and it possesses a variety of carbohydrate substitutions and non-carbohydrate decorations which can impede enzymatic degradation by glycoside hydrolases. Carbohydrate esterases are able to cleave the ester-linked decorations and thereby improve the accessibility of the xylan backbone to glycoside hydrolases, thus improving the degradation process. Enzymes comprising multiple catalytic glycoside hydrolase domains on the same polypeptide have previously been shown to exhibit intramolecular synergism during degradation of biomass. Similarly, natively fused carbohydrate esterase domains are encoded by certain bacteria, but whether these enzymes can result in similar synergistic boosts in biomass degradation has not previously been evaluated. Results Two carbohydrate esterases with similar architectures, each comprising two distinct physically linked catalytic domains from families 1 (CE1) and 6 (CE6), were selected from xylan-targeting polysaccharide utilization loci (PULs) encoded by the Bacteroidetes species Bacteroides ovatus and Flavobacterium johnsoniae. The full-length enzymes as well as the individual catalytic domains showed activity on a range of synthetic model substrates, corn cob biomass, and Japanese beechwood biomass, with predominant acetyl esterase activity for the N-terminal CE6 domains and feruloyl esterase activity for the C-terminal CE1 domains. Moreover, several of the enzyme constructs were able to substantially boost the performance of a commercially available xylanase on corn cob biomass (close to twofold) and Japanese beechwood biomass (up to 20-fold). Interestingly, a significant improvement in xylanase biomass degradation was observed following addition of the full-length multidomain enzyme from B. ovatus versus the addition of its two separated single domains, indicating an intramolecular synergy between the esterase domains. Despite high sequence similarities between the esterase domains from B. ovatus and F. johnsoniae, their addition to the xylanolytic reaction led to different degradation patterns. Conclusion We demonstrated that multidomain carbohydrate esterases, targeting the non-carbohydrate decorations on different xylan polysaccharides, can considerably facilitate glycoside hydrolase-mediated hydrolysis of xylan and xylan-rich biomass. Moreover, we demonstrated for the first time a synergistic effect between the two fused catalytic domains of a multidomain carbohydrate esterase.

Gross calorific value estimation for milled maize cob biomass using near infrared spectroscopy

The maize cob biomass is one of important biomass crops in Thailand. Nowadays, the use of the biomass as renewable resource is increasing, especially residue agriculture waste. As we know that the biomass properties impact combustion, in order to achieve boiler efficiency, its energy characteristics of biomass was required immediately before burning. This work uses the FT-near infrared spectroscopy to estimate gross calorific value (GCV) of maize cob as the rapid method. Each sample was scanned using diffuse reflectance mode at a wavenumber range between 12500-3600 cm-1. The scanning was done with a resolution of 8 cm-1 and completed 32 scans per sample, then averaged to be one spectrum. The results showed that this technique could decrease a processing time to 1-2 minutes per sample to determine GCV whereas alternatively the current method used a processing time of 25-30 minutes per sample. The capacity of the model gave root mean square error of cross validation (RMSECV) of 91.1 Jg-1, which was low. Hence, the model was acceptable and cloud be used for screening.